D52 tumor proteins (TPD52) are frequently over-expressed in cancer and malignancies. They are multifunctional proteins, althoughtheir precise role(s) in cancer have not been clear. The proposed research has been designed to solve high resolution structures of TPD52 proteins and their complexes with endogenous binding partners. The long-term goal of this research is to elucidate and control the molecular mechanisms of TPD52 activities in physiological and pathological processes. Specifically, we propose to elucidate the molecular architecture of this class of proteins, and the structural basis of their interaction with their binding partners, Annexin VI and Mal2. The primary impact of our anticipated findings would be improved chances of targeting the structure and function of TPD52 proteins with small molecules intended ultimately for cancer treatment. Standard DNA cloning methods will be employed to obtain affinity-tagged expression vectors of the TPD52 proteins, MAL2 protein and annexin VI, for different expression systems.

We will first over-express the proteins in E. coli (or in yeast or insect cells if necessary) as Histag or Glutathione S-Tranferase fusions proteins. Combinations of affinity-, ion exchange- and size exclusion chromatography will be used to further purify the protein to homogeneity. The hanging drop vapor-diffusion technique will be employed to screen a large number of conditions for crystallization using commercial kits. Once any leads have been identified, the conditions will be optimized for crystal growth. The crystals will be evaluated by an Raxis VI++ imaging plate or synchrotrons. Diffraction data sets will be collected and analyzed with various software packages. The Multiple Isomouphous Replacement and the Multiple Amormalous Scattering techniques will be used to solve the structures of the novel proteins. Molecular Replacement techniques will be employed if a structure similar to our protein becomes available. The structures will be refined with CNS and the refined structures will be compared with all other availablestructures in the Protein Data Bank in order to determine the structural family that the protein belongs to. The human health significance of this research is that once the complex structures of the TPD52 proteins are known, it should be possible to shed light on the roles of the TPD52 proteins in cancer, design ways to control or interfere the TPD52 functions, and help to find new ways for cancer therapy.